SNAP-25a and -25b isoforms are both expressed in insulin-secreting cells and can function in insulin secretion

摘要

pThe tSNARE (the target-membrane soluble NSF-attachment protein receptor, where NSF is iN/i-ethylmaleimide-sensitive fusion protein) synaptosomal-associated protein of 25 kDa (SNAP-25) is expressed in pancreatic B-cells and its cleavage by botulinum neurotoxin E (BoNT/E) abolishes stimulated secretion of insulin. In the nervous system, two SNAP-25 isoforms (a and b) have been described that are produced by alternative splicing. Here it is shown, using reverse transcriptase PCR, that messages for both SNAP-25 isoforms are expressed in primary pancreatic B and non-B cells as well as in insulin-secreting cell lines. After transfection, both isoforms can be detected at the plasma membrane as well as in an intracellular perinuclear region in the insulin-secreting cell line, HIT. To test for the functional role of the two isoforms in insulin secretion, mutant forms of SNAP-25a and b resistant against cleavage by BoNT/E were generated. Such mutant SNAP-25, when expressed in HIT cells, is not inactivated by BoNT/E and its ability to restore insulin secretion can thus be investigated. To obtain the toxin-resistant mutant isoforms, the sequence around the BoNT/E cleavage site (Rsup176/supQIDRIMsup182/sup) was changed to Psup176/supQIKRITsup182/sup. This is the sequence of the equivalent region of human SNAP-23 (Psup187/sup–Tsup194/sup), which has been shown to be resistant to BoNT/E. The mutant SNAP-25 was resistant to BoNT/E iin iv/iitro/i and iin iv/iiiv/io/i and both mutant isoforms were able to reconstitute insulin secretion from toxin-treated HIT cells./p